Project Summary As obligate cellular parasites, viruses employ a variety of strategies to co-opt the host cell?s machinery, using it to generate the molecules needed for successful infection. Many of these strategies involve viral RNA that forms specific structures able to interact with and manipulate cellular components. An important example is found in the mosquito-borne flaviviruses, which co-opt a cellular exoribonuclease and use it to generate pathogenically-important non-coding RNAs. Specifically, the 5??3? exoribonuclease Xrn1 is recruited to the genomic RNA, processively degrades it, but then halts at specific locations in the genome. This programmed ?exoribonuclease resistance? depends on specific three-dimensional RNA structures that are embedded in the flaviviral RNA. The exoribonuclease-resistant RNAs (xrRNAs) of the mosquito-borne flaviviruses are the prototypes of this process and we have learned much by studying them. However, it is now clear that the strategy of co- opting and exploiting cellular exoribonucleases is not limited to these viruses, but may be widespread. Evidence suggests that diverse viruses use different types of exoribonuclease-resistant RNA elements as a means to process long precursor RNAs into shorter, biologically active RNAs. However, despite the emerging importance of these novel exoribonuclease- resistant RNA structures and the mechanisms they perform, we know almost nothing about them. Among the burning fundamental questions: Do all of these putative Xrn1-resistant elements use a similar mechanism? Despite no obvious sequence similarity, are they all folded RNAs? Are they all RNA structure-driven, or do some require bound proteins? Are the folds of these different RNAs similar, or has nature evolved many ways to achieve the goal of blocking progression of an exoribonuclease? Our understanding of these processes in diverse viruses is hampered by a lack of basic information about various xrRNA structures. The focus of this proposal is therefore to drive the field forward by studying several unexplored examples of xrRNAs. We aim to gain insight into the breadth and diversity of the exoribonuclease resistance phenomenon, to discover fundamental principles of exoribonuclease resistance that may be applicable across the larger viral world, and to develop new technology to enable us to find or predict exoribonuclease structures in other viruses and contexts. We propose three aims: (1) Determine the essential sequences, structural determinants, and mechanistic characteristics of exoribonuclease resistance by a diverse set of flaviviral RNAs. (2) Define sequences and structures of RNAs from the Dianthoviruses and Rift Valley Fever Virus that confer exoribonuclease resistance, and (3) Develop a synthetic expanded phylogeny of Xrn1-resistant RNAs and use this to computationally search for unidentified resistant RNAs in other viruses. Our approach is to combine biochemical assays that are unique to our lab and that comprise a comprehensive set of tools for exploring these RNAs, structural biology to include x-ray crystallography, and in vitro selections coupled with computational tools. The research described here will contribute significant basic knowledge regarding an important molecular process of broad applicability to viral disease, a necessary step between the discovery of a mechanism and the targeting of it for therapeutic intervention.